The dengue virus genome codes for a polyprotein in the order of NH2-C-PreM- E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH that is proteolytically cleaved to generate the 10 individual proteins. Our previous studies indicated that cleavage between NS2B and NS3 is critical for the functional activity of the viral proteinase. Our goal is to elucidate the mechanism of polyprotein processing and apply the information to engineer stable dengue virus mutants that exhibit restriction of viral replication because a subset of such mutants should be less virulent than wild-type virus but still able to achieve a satisfactory level of immunogenicity during infection. This project was initiated to investigate the sequence requirements for proteolytic processing of dengue virus NS2B-NS3. Deletion analysis showed that two amino acids upstream of the NS2B-NS3 junction are sufficient for cleavage to take place. Subsequently, mutants of NS2B-NS3 containing single amino acid substitutions at the cleavage junction were constructed for analysis of their cleavage phenotype. Several substitutions resulted in an intermediate or a low level of cleavage. The finding that mutants of NS2B-NS3 containing substitutions at the cleavage junction exhibited a reduction of cleavage efficiency has provided a body of information for molecular analysis such as engineering of dengue virus mutants that are restricted in growth in a cell culture and perhaps in infected animals. Finally, full-length dengue type 4 cDNA constructs containing single amino acid substitutions at the NS2B-NS3 cleavage junction were prepared and used as templates for in vitro transcription. Recovered dengue virus mutants were verified for the presence of the mutant sequence in genomic RNA. Work is in progress to analyze the growth phenotype of these mutants. A series of deletion constructs of NS2A or NS2B that has been shown to affect poly protein processing will be employed to construct deletion mutants of dengue virus. Deletion mutants should be less subject to reversion of phenotype than amino acid substitution mutants. Future plans will include evaluation of virulence and immunogenicity of these mutants in infected animals.